U.S. patent number 5,744,042 [Application Number 08/570,816] was granted by the patent office on 1998-04-28 for method for the separation of protein-bound substances from a protein-containing liquid by dialysis.
Invention is credited to Steffen Mitzner, Wolfgang Ramlow, Jan Stange.
United States Patent |
5,744,042 |
Stange , et al. |
April 28, 1998 |
Method for the separation of protein-bound substances from a
protein-containing liquid by dialysis
Abstract
The present invention provides a process of separating
substances bound to a first protein in a first liquid by means of a
membrane separating the first liquid from a second liquid, the
process comprising the steps of: a) impregnating the membrane by
passing along the membrane a solution comprising a first acceptor
protein having an acceptor function for the substances to be
separated; b) dialyzing the first liquid against the second liquid,
the second liquid comprising a second acceptor protein having an
acceptor function for the substances to be separated, wherein the
membrane separating the first and second liquids contains
tunnel-like structures that permit passage of the substances to be
separated to the second liquid but exclude passage of the first
protein and the second acceptor protein.
Inventors: |
Stange; Jan (18055 Rostock,
DE), Mitzner; Steffen (18055 Rostock, DE),
Ramlow; Wolfgang (D-18209 Bad Doberan, DE) |
Family
ID: |
25924257 |
Appl.
No.: |
08/570,816 |
Filed: |
December 12, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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123002 |
Sep 17, 1993 |
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Current U.S.
Class: |
210/645; 210/644;
210/646; 436/177; 436/178 |
Current CPC
Class: |
A61M
1/1654 (20130101); A61M 1/1696 (20130101); B01D
61/243 (20130101); B01D 69/02 (20130101); B01D
69/141 (20130101); A61M 1/3475 (20140204); Y10T
436/25375 (20150115); Y10T 436/255 (20150115) |
Current International
Class: |
A61M
1/16 (20060101); B01D 69/02 (20060101); B01D
69/14 (20060101); B01D 61/24 (20060101); B01D
69/00 (20060101); A61M 1/34 (20060101); B01D
061/24 () |
Field of
Search: |
;210/638,644,645,646,650,651,500.23,502.1,632 ;436/177,178
;530/362,363,413,417 ;435/180,182 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Kim; John
Attorney, Agent or Firm: Dressler, Rockey, Milnamow &
Katz, Ltd.
Parent Case Text
This is a continuation, of application Ser. No. 08/123,002, filed
Sep. 17, 1993, now abandoned.
Claims
What is claimed is:
1. A process of separating substances bound to a first protein in a
first liquid by means of a semipermeable membrane separating the
first liquid from a second liquid, the process comprising the steps
of:
a) impregnating the membrane by passing a solution along both sides
of the membrane, the solution containing the first protein, which
first protein has an acceptor function for the substances to be
separated and an affinity for the membrane, for a period of time
sufficient to permit penetration and adsorption of the first
protein on both sides of the membrane; and
b) passing the first liquid and the second liquid on each side of
said semipermeable membrane, the second liquid containing the first
protein in free form, wherein the membrane contains (i) a first
structure facing the first liquid and having tunnels with a length
of 0.01 to 0.1 micrometer and a diameter that permits passage of
the substances to be separated but excludes passage of the first
protein and (ii) a port- and adsorption structure facing the second
liquid that permits the second liquid and the first protein therein
to pass into and out of the port- and adsorption structure, whereby
the substances bound to the first protein in the first liquid are
separated from the first protein and passed through the membrane to
the first protein in the second liquid.
2. The process of claim 1 wherein membrane material is selected
from the group consisting of polysulfones, polyamides,
polycarbonates, polyesters, acrylonitrile polymers, vinyl alcohol
polymers, acrylate polymers, methacrylate polymers and cellulose
acetate polymers.
3. The process of claim 2 wherein the membrane material is
polysulfone.
4. The process of claim 1 wherein the first liquid is plasma or
blood and the second liquid comprises human serum albumin as a
protein having an acceptor function for the protein-bound
substances.
5. The process of claim 4 wherein the first protein is human serum
albumin.
6. The process of claim 1 wherein the second liquid comprises human
serum albumin.
7. The process of claim 6 wherein the human serum albumin is in a
concentration from about 6 to about 40 grams per 100 ml.
8. The process of claim 6 wherein the human serum albumin is in a
concentration from about 8 to about 30 grams per 100 ml.
9. The process of claim 6 wherein the human serum albumin is in a
concentration from about 8 to about 20 grams per 100 ml.
10. The process of claim 1 wherein the second liquid is transported
with an alternating influx and outflux movement in a direction
perpendicular to an outer membrane surface such that the second
liquid moves into and out of the port- and adsorption
structure.
11. The process of claim 1 wherein impregnating is accomplished at
a temperature of from about 15.degree. C. to about 40.degree. C., a
pH of from about 5 to about 9 and for a time from about 1 to about
120 minutes.
Description
1. TECHNICAL FIELD
The present invention relates to membranes and membrane transport
processes, and in particular to an effective method for the
separation of undesired or potentially harmful protein-bound
substances (PBS) and, if present, water-soluble substances
(low-molecular weight and middle-molecular weight substances)
and/or lipophilic substances, from a protein-containing liquid such
as plasma and blood by dialysis using said membranes.
2. Background
The separation of substances which are strongly bound to a valuable
protein from that protein by dialysis is impossible or at least
accompanied with a number of difficulties. This is especially
relevant if the protein is contained in a complex mixture such as
blood or plasma.
In medicine separation techniques are widely accepted for the
separation of hydrophilic toxins from blood e.g. in end stage renal
disease (ESRD, uremia). Today, maintenanced hemodialysis (HD) is
the live-saving long-term treatment of choice in ESRD patients if
no kidney transplantation is available. However, the removal of
protein-bound or lipophilic toxins remains, at present, an unsolved
problem in medicine. Especially albumin-bound toxins (ABT) have
been shown to be involved in the pathogenesis of different
exogenous and endogenous intoxications in patients.
Thus ABTs such as butyric, valeric, caproic and caprylic acid,
thyroxine and tryptophane, unconjugated bilirubin, mercaptans,
digoxin-like immunoreactive substances, benzodiazepine-like
substances, aromatic amino acids and phenols seem to be responsible
for the induction of hepatic encephalopathy and cerebral edema in
fulminant hepatic failure (FHF).
These substances are assumed to be at least partially responsible
for lethal cerebral alterations seen in FHF. Moreover, FHF is
typically accompanied by coagulopathy, defective ventilation,
hypoxemia, pulmonary edema, electrolyte imbalance, acid-base
disturbances, renal dysfunction, hypoglycemia, cardiac dysfunction,
sepsis, hemorrhage leading to multiorgan failure and death. The
mortality has been on an unchanged high level between 60 and 100%
since years, depending on e.g. the age of the patient and the stage
of encephalopathy at hospital entry.
In chronic liver insufficiency the same group of ABTs accumulates
due to the insufficient metabolism of the liver in the blood and
brain although the primary toxins may be different (e.g. ethanol).
Therefore, symptoms such as encephalopathy normally seen in FHF may
occur in these patients, too, and acute exacerbations mimicking the
picture of FHF are seen frequently.
Newborn hyperbilirubinemia is another clinically relevant
endogeneous intoxication. This form of hyperbilirubinemia is known
to have damaging effects to the newborn brain because of the
immature blood-brain barrier. Toxic effects of bilirubin in the
adult human are under discussion. At least in very high
concentrations the toxic effect could be clearly demonstrated.
Furthermore, there is a large number of drugs known to have a
highly albumin-binding rate in cases of accidental overdosage or
suicidal intoxications by e.g. tricyclic antidepressants, digoxin,
digitoxin, theophylline or a benzodiazepine.
While the importance of ABT in the above-mentioned cases is
generally acknowledged, there is also a significant accumulation of
albumin-bound toxins in ESRD, e.g. furancarboxylic acids, indoxyl
sulfate, aluminum ions, phenols and other organic acids, although
patients are on regular HD treatment. The unsatisfactory search for
"uremic toxins" among the "middle molecules" spectrum on the one
hand and the fact that uremic plasma shows more toxic effects than
its ultrafiltrate on the other hand led to the assumption that some
of the "uremic toxins" may be also protein-bound.
Meanwhile this theory which has its origin as early as in 1976 has
been accepted by the American Society for Artificial Internal
Organs (1993) and many specialists in the USA, Europe and Japan.
This indicates that not only the liver but also the kidneys take
part in the separation of albumin-bound substances from the blood
in order to keep them at non-toxic levels. This is already known
for some drugs and indicator substances (e.g. phenol red) and has
been shown recently also for furan derivatives as a special
excretoric function of the kidneys which cannot adequately be
replaced by hemodialysis. This explains that in dialysis patients
the concentrations of these molecules reach levels which may be of
pathophysiological relevance. Underlining this it has been shown
that furancarboxylic acids accumulating in chronic hemodialysis
patients are strong inhibitors of mitochondrial cell respiration.
Furthermore, furan derivatives act as inhibitors of cell
proliferation by interfering with DNA synthesis as shown in
vitro.
In vivo relevant long-term complications of maintenanced HD
treatment in ESRD are seen in almost every patient (e.g.
dialysis-associated anemia, suppressed immune response, high
infection rates, encephalopathy) that are caused by insufficiency
of highly specialized or proliferative tissues (e.g. blood forming
tissue). Suggesting a relation between these in vitro and in vivo
results clinical investigations have shown that e.g. the grade of
anemia in hemodialyzed patients strongly correlates with the plasma
levels of furancarboxylic acids.
All states of disease mentioned above have in common not only the
ABTs involved in the pathogenesis but also very high treatment
costs for either intensive care and transplantation or long-term
treatment with supplementation therapy (e.g. erythropoietin) and
repeating hospital stays. Therefore, ABT-associated diseases have a
considerable economic dimension with high patient numbers and
treatment costs on the one hand and a bad overall prognosis leading
to death or invalidity on the other hand (e.g. 400,000 patients on
maintenanced HD worldwide with approximately 60 million treatments
per year).
Treatment of ABT-associated diseases--State of the art
1. Dialysis and Ultrafiltration
Because of their affinity to the non-dialyzable albumin molecule
ABTs are not able to permeate to a significant degree into the
dialysate liquid in vitro and in vivo.
Moreover, many of these ABTs are highly lipophilic and not soluble
in water.
Conventional hemodialysis effectively separates low molecular
weight substances (<1500 daltons) from plasma but no
protein-bound substances which are believed to be responsible for
the detrimental effects of liver insufficiency. Hemodialysis using
e.g. large-pore polyacrylonitrile membranes effectively removes
middle molecular weight substances (1500-5000 daltons) in addition
to the low molecular weight substances but again no protein-bound
substances in experimental animals; see De Groot, GH, Schalm SW,
Schicht I: Large-pore hemodialytic procedures in pigs with ischemic
hepatic necrosis; a randomized study, Hepatogastroenterol. Vol. 31
(1984), 254; De Groot, GH, Schalm SW, Schicht I: Comparison of
large-pore membrane hemodialysis and cross-dialysis in acute
hepatic insufficiency in pigs, Eur. J. Clin. Invest., Vol. 13
(1983), 65.
Therefore, dialysis and ultrafiltration are not satisfactory
methods for the separation of these substances.
2. Peritoneal dialysis
Patients undergoing peritoneal dialysis (PD) as an alternative
treatment lose up to 10% of the total serum albumin during a single
PD session and by this way, of course, also toxic albumin-bound
substances (ABT). In clinical studies typical long-term
complications of HD patients, especially the above-mentioned
anemia, could not be observed in PD patients. However, this
enhanced removal of albumin-bound toxins by PD is accompanied by
the loss of albumin which correlates with an increased mortality.
Furthermore, PD is associated with a high risk of peritonitis.
3. Erythropoietin therapy
Erythropoietin therapy partially reverses the
dialysis/uremia-associated anemia but is expensive. The reason for
the anemia is not a lack of endogenous erythropoietin. Arterial
hypertension is one of the most often seen adverse effects in this
therapy.
4. Kidney transplantation
This method can ameliorate the problem of insufficient removal of
ABTs in ESRD. Nevertheless, it has a number of severe complications
including operation risk, intensive immunosuppression, high tumor
risk. The overall life expectancy is not different from HD
treatment.
5. Hemo-/Plasmaperfusion
Hemo- or plasmaperfusion over different or other adsorbents such as
ion exchange resins and charcoal are also effective but not
sufficiently specific. These methods also remove essential
substances like hormones (corticosteroids, thyroxine) which are
linked to their own transport proteins (corticosteroid transporting
protein or thyroxine transporting protein).
In most of the studies made in this regard blood was perfused
through sorbent columns containing charcoal or ion exchange resins.
By this method protein-bound toxins were removed effectively but
this was accompanied by the following disadvantages:
a) loss of blood cells, especially platelets
b) loss of clotting factors
c) activation of blood cells (platelets, leukocytes)
d) activation of the complement system
e) loss of essential plasma components blood compounds (hormones,
vitamines, growth factors) due to the absolutely unspecific mode of
binding
f) release of microparticles, especially when charcoal adsorbents
were used.
In order to attenuate the adverse effects seen in charcoal
perfusion charcoal particles are coated with different
blood-compatible substances but these charcoal adsorbents exhibit a
decreased ability to bind protein-bound substances. Hemoperfusion
over micro-encapsulated charcoal effectively removes low-molecular
weight substances (<1500 daltons) from plasma but no
protein-bound substances which are believed to be responsible for
the detrimental effects of liver insufficiency.
Of all extracorporeal dialysis and adsorption techniques especially
hemoperfusion over an albumin-coated resin (e.g. Amberlite XAD-7)
or agarose beads to which albumin had been covalently linked have
been shown to effectively remove bilirubin and other toxic
protein-bound organic anions from the blood of experimental animals
(Wilson R. A., Webster K. H., Hofmann A. F.: Toward an artificial
liver: in vitro removal of unbound and protein-bound plasma
compounds related to hepatic failure, Gastroenterol., Vol. 62
(1972), 1191.; Scharschmidt B. F., Plotz P. H., Berk P. D.:
Removing substances from blood by affinity chromatography. II.
Removing bilirubin from blood of jaundiced rats by hemoperfusion
over albumin-conjugated agarose beads, J. Clin. Invest., Vol. 53
(1974), 786. No assessment was made concerning the selectivity or
possible adverse effects of this method.
However, these albumin-coated adsorbents did not remove low and
middle molecular weight substances (Zakim and Boyer (ed.),
Hepatology. A textbook of liver disease, W. B. Saunders Company,
1990, p. 479-482).
In conclusion hemo- or plasma-perfusion remain an unselective
method.
6. Plasma exchange
In this method the plasma of the patient is separated from blood by
filtration or by centrifugation and is replaced by an albumin
solution or plasma. This procedure shows the following
disadvantages:
a) most protein-bound toxins are not only distributed in plasma but
also in tissues. After a plasma exchange session a redistribution
of the toxins into the purified plasma occurs. This was accompanied
with the clinical observation that patients in FHF undergoing this
treatment awoke from coma but only for the time of treatment and
fell back into deep coma after the end of treatment. Thus, plasma
exchange had to be done frequently to be effective, which led to
typical immunological reactions mentioned below.
b) the observed immunological reactions arising from frequent
plasma exchange have been known for a long time from blood
transfusions as allergic reactions up to anaphylactoid reactions. A
special manifestation was described as the "transfusion lung".
c) the risk of viral infection is present (HIV, hepatitis).
d) in FHF the organism answers the massive cell degradation with an
increased secretion of growth hormones in order to enhance organ
regeneration. These growth hormon levels are higher than in healthy
persons and are therefore not present in the donor plasma.
e) plasma exchange is expensive.
7. Exchange transfusions
In severe newborn-hyperbilirubinemia today exchange transfusions
are the treatment method of choice. The therapy is helpful to the
patient because irreversible brain damage is prevented. However,
the transfusions have similar disadvantages as mentioned above for
plasma exchange.
8. Other extracorporeal methods
Different other approaches especially towards the field of FHF
treatment and with respect to the removal of protein-bound toxins
have been made in the past years:
a) charcoal-impregnated hollow fibers/dialysis against sorbent
suspensions
Trials to combine the hemocompatibility of dialysis with the
effectivity of charcoal adsorption by filling charcoal into
cellulosic membranes or recirculate adsorbent suspensions (powdered
charcoal and ion exchange resins) in order to remove "middle
molecules" did not at all or not sufficiently come up to the
expectations. Especially the removal of strongly protein-bound
fractions of ABT (e.g. unconjugated bilirubin) was insufficient.
The reasons for this, in our opinion, was the use of membranes
which lack the predispositions for the "tunneling effect" which
will be described below.
b) lipophilic liquid membranes
A further effort was made to remove protein-bound toxins by the use
of hydrophobic semipermeable polymer membranes filled with
lipophilic liquids enabling the passage of water-insoluble
compounds like middle-molecular weight fatty acids and mercaptans.
An alkaline dialysate (pH 13) solution was proposed as acceptor
solution for these toxins. The disadvantages of this method are the
following:
1. Some of the ABTs are insoluble in this liquid membrane and are
therefore excluded from the removal process (e.g. unconjugated
bilirubin).
2. The insolubility of hydrophilic toxins (e.g. ammonia) in the
liquid phase excluded their removal, too, and required the
additional use of a further dialysis procedure, because most of the
above described intoxications were of complex nature including
hydrophilic and hydrophobic toxins. By the use of an additional
dialysis device the extracorporeal blood volume as well as
blood-polymer contact increases. Last but not least costs are very
high.
3. The use of alkaline dialysate solutions might be of potential
harm to the patient influencing the endogenous pH value at least in
cases of membrane damage.
9. Biohybrid systems
These systems are bioreactors designed for the processing of blood
or plasma which include biological components (e.g. liver slices,
liver cells) which will provide a natural detoxification. Because
of the limited functionality and short life of liver slices only
cell-based systems are acceptable. The disadvantages of this method
are:
a) there is no stable and save source providing sufficient amounts
of cells needed for the treatment.
The most important cell for the detoxification of protein-bound
toxins, the hepatocyte of the liver until today could not be
produced by cultivation in vitro without genetic manipulation which
is followed by cell alteration and therefore connected with further
risks that cannot be calculated. The use of tumor liver cells which
could be produced by in vitro cultivation is connected with the
risk of developing tumor and furthermore with risks arising from an
metabolic alteration of the cells which cannot be foreseen. The use
of animal cells is connected with immunological reactions between
the plasma/blood of the patient on the one hand and the xenogenic
liver cell on the other.
b) difficulty of the method
Because of the poor possibilities for cryoconservation of the cells
or complete bioreactors the preparation of the device has to be
done always just prior to use. This makes this method usable only
for highly specialized centers.
c) high costs
All steps of this treatment involve high costs.
10. Liver transplantation
Today this is the method of choice in patients with either chronic
or acute end stage liver failure because it shows the best long
time survival rates from all therapies mentioned for liver failure.
The limiting factors of this method are limited availability of
suitable donor organs (long waiting lists), surgery risk, life-long
immune suppressive therapy to avoid rejection associated with the
common complications (e.g. high infection risk, high tumor risk,
risk of diabetes). Moreover, the indications for transplantation
are very tight, automatically excluding a large number of fatally
ill patients. Thus, transplantation did not avoid the need for
sufficient detoxification procedures for those patients not
eligible for surgery. A number of those patients eligible for
surgery is in a status too bad to undergo surgery because of FHF
with coma, hypotension, high bleeding risk. These patients are in
need for a sufficient "liver bridging treatment" to prepare for
transplantation and, in a number of cases, when patients come into
FHF after transplantation it is needed again. Last but not least
transplantation causes high costs arising from surgery as well as
from the immune supressive therapy.
Therefore, it would be useful to provide a method for the
separation of protein-bound substances such as ABTs from
protein-containing liquids, e.g. from blood or plasma of patients
suffering e.g. from FHF, chronic liver insufficiency, accidental or
suicidal drug overdosage and ESRD, which is effective, safe and
less expensive. It would be advantageous if this method did not
interfere with the homeostasis of the liquid, e.g. blood, for
instance, by undesired removal of valuable components, e.g.
proteins, from the liquid or addition of potentially harmful
(toxic) substances to the liquid.
Thus, one object of the present invention is to provide a method
for the separation of protein-bound substances from a
protein-containing liquid containing these substances by dialysis.
Another object of the invention is to provide a membrane for the
separation of said protein-bound substances from a
protein-containing liquid containing these substances by
dialysis.
Further objects will become apparent from the following
description, drawings and claims.
SUMMARY OF THE INVENTION
The invention is based on the unexpected finding that protein-bound
substances (PBS), including even those being strongly bound, can be
removed from protein-containing liquids (A) by dialysis against a
dialysate liquid (B) and by means of a semipermeable membrane and
by means of a protein having an acceptor function for the PBS.
Preferably, the dialysate liquid (B) of the present invention
contains a protein having an acceptor function for the
protein-bound substance (PBS) to be removed from the
protein-containing liquid (A), in general of the type of protein
present in the PBS-protein complex in liquid (A).
In case of plasma or blood as protein-containing liquid (A) the
preferred acceptor protein in the dialysate liquid (B) is albumin,
in particular human serum albumin or recombinant human albumin.
The membrane of the present invention preferably comprises two
functionally different parts. One part has the actual separating
membrane function permitting the PBS and, if present, the
water-soluble substances to pass through under the conditions of
the process of the present invention and excluding the protein(s)
which had bound the PBS in liquid (A) and the acceptor protein of
liquid (B), and the other part has a port- and adsorption function.
Preferably, the membrane is coated with a protein having an
acceptor function for the PBS. In a preferred embodiment the
membrane of the present invention comprises a tunnel-like structure
facing the liquid (A) side, the tunnels having a length less than
about 10 .mu.m and having a diameter sufficiently small to exclude
the protein in liquid (A), and a port- and adsorption-structure on
the dialysate liquid (B) side. Preferably, the membrane is coated
on at least one side, preferably the dialysate liquid (B) side,
with a thin film of a protein having an acceptor function for the
protein-bound substances.
The membrane of the present invention may have the macroscopic form
of a flat film, a thin-walled but large diameter tube, or
preferably fine hollow fibers. Membrane technology, hollow-fiber
membranes, and dialysis is described in Kirk-Othmer, Encyclopedia
of Chemical Technology, third edition, Vol. 7 (1979), 564-579, in
particular 574-577, Vol. 12 (1980), 492-517 and Vol. 15 (1981),
92-131. Furthermore, membranes and membrane separation processes
are described in Ullmann's Encyclopedia of Industrial Chemistry,
Fifth edition, Vol A 16 (1990), 187-263.
The matrix material for the membrane may be made from many
materials, including ceramics, graphite, metals, metal oxides, and
polymers, as long as they have an affinity towards the protein on
the liquid (A) and the dialysate liquid (B). The methods used most
widely today are sintering of powders, stretching of films,
irradiation and etching of films and phase inversion techniques.
The preferred materials for the membranes of the present invention
are organic polymers selected from the group consisting of
polysulfones, polyamides, polycarbonates, polyesters, acrylonitrile
polymers, vinyl alcohol polymers, acrylate polymers, methacrylate
polymers, and cellulose acetate polymers. Especially preferred are
polysulfone membranes hydrophilized with e.g.
polyvinylpyrrolidone.
A precise and complete definition of a membrane is rather
difficult; see Ullmann, loc. cit., page 190-191, No. 2.1 and 2.2. A
membrane can be homogeneous, microporous, or heterogeneous,
symmetric or asymmetric in structure. It may be neutral, or may
have functional groups with specific binding or complexing
abilities. The most important membranes currently employed in
separation processes are the asymmetric membranes; see Ullmann,
loc. cit., page 219 et seq., No. 4.2. Known asymmetric membranes
have a "finger"-type structure, a sponge-type structure with a
graded pore size distribution or a sponge type structure with a
uniform pore size distribution; see Ullmann, loc. cit., page
223-224.
The most preferred membrane structure of the present invention is
an asymmetric membrane composed of a thin selective skin layer of a
highly porous substructure, with pores penetrating the membrane
more or less perpendicularly in the form of fingers or channels
from the skin downward. The very thin skin represents the actual
membrane and may contain pores. The porous substructure serves as a
support for the skin layer and permits the protein having an
acceptor function to come close to the skin and to accept the
protein-bound substances penetrating the skin from the liquid (A)
side towards the dialysate liquid (B) side.
Prior to the separation procedure the membrane is preferably
prepared as follows. The membrane is treated from the liquid (A)
side and/or from the liquid (B) side with a liquid which contains
the protein having an acceptor function, preferably a 0.9% NaCl
solution, containing the acceptor protein, preferably human serum
albumin in a concentration from about 1 to about 50 g/100 ml, more
preferably from about 5 to about 20 g/100 ml. The treatment time is
about 1 to about 30 min, preferably about 10 to about 20 min, at a
temperature from about 15.degree. to about 40.degree. C.,
preferably from about 18.degree. to about 37.degree. C.
The method of the present invention for the separation of
protein-bound substances and, of course conventional water-soluble
substances that may be present, from a protein containing liquid
(A) is carried out as follows:
The liquid (A) to be purified is passed through a dialyzer
comprising a membrane along the liquid (A) side of the membrane
with a flow rate of about 50 to about 500 ml/min, preferably about
100 to about 200 ml/min per one sqm membrane area on the liquid (A)
side. The dialysate liquid
(B) is passed along the dialysate liquid (B) side of the membrane
with a flow rate of about 50 to about 500 ml/min, preferably of
about 100 to about 200 ml/min per one sqm membrane area and
preferably with the same flow rate as the liquid (A).
The dialysate liquid (B) obtained and containing the protein-bound
substances and possibly water-soluble substances from liquid (A)
preferably is then passed through a second conventional dialyzer
that is connected to a conventional dialysis machine. A dialysis
against an aqueous standard dialysate is carried out. By this
dialysis water-soluble substances are exchanged between the
dialysate liquid (B) and the standard dialysate. Thus,
water-soluble toxins such as urea or creatinine can be separated
from the dialysate liquid (B) and electrolytes, glucose and pH can
be balanced in the dialysate liquid (B) and, therefore, also in
liquid (A). The dialysate liquid (B) obtained freed from
water-soluble substances preferably is then passed through a
charcoal-adsorbent, e.g. Adsorba 300 C from GAMBRO AB or N350 from
ASAHI, and an anion exchange column, e.g. BR350 from ASAHI, to
remove the protein-bound substances from the protein acceptor in
the dialysate liquid (B). The purified dialysate liquid (B)
obtained is then returned to the dialysate liquid (B) side of the
membrane of the present invention and reused.
Other advantages and benefits will be apparent to those skilled in
the art from the detailed description that follows.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the results of an in vitro separation of
protein-bound substances (unconjugated bilirubin, free fatty acids,
phenol, sulfobromophthalein) from plasma (liquid (A)) in accordance
with the method of the present invention. The decrease of the
protein-bound substances (PBS) is shown in percent of the initial
concentrations in the plasma (liquid (A)).
FIG. 2 is a graph showing the increase of protein-bound substances
in dialysate solution (liquid (B); corresponding to FIG. 1 and
obtained from the same experiment) in percent of the initial
concentrations in the plasma (liquid (A)).
FIG. 3 is a graph showing total serum bilirubin concentrations in a
29 year old patient with an acute exacerbation of a chronic liver
insufficiency during a 4 days period of MARS treatment followed by
a 2 days conventional HDF.
FIG. 4, 5 and 6 illustrate embodiments for the purification of
blood by means of hemodialysis according to the present
invention.
______________________________________ DA Dialyzer A (albumin
coated Membrane) DB Dialyzer B (convertional dialyzer) ADS A
Adsorbent A ADS B Adsorbent B Pump 1 Blood roller pump Pump 2 first
albumin dialysate roller pump Pump 3 second albumin dialysate
roller pump I-V Safety devices: I Blood line bubble snare II
Albumin dialysate line bubble snare III Blood lack detector IV
Veneous drop chamber V Safety clamp in/out Blood inflow and outflow
X/Y Albumin dialysate flow connectors (DA) U/V Conventional
dialysate flow connectors (DB)
______________________________________
The blood (liquid (A)) flows from "in" pumped by pump 1 through the
bubble snare into the dialyzer (DA) and after passing it through
the veneous drop chamber, after that passing the safety clamp.
Safety devices are not part of the invention but have to be used
because of safety regulations. The albumin dialysate (liquid (B))
is pumped by roller pump 2 from connector X in direction to
connector Y or from Y to X, but preferably counter-currently to the
blood flow, passing at first the blood lack detector (III) and also
a bubble snare (II). Thereafter, liquid (B) passes a conventional
dialyzer (DB) and, if desired, adsorbent columns (A and B).
Other possibilities are:
The liquid (B) is only passed through adsorbents and not through a
dialyzer
The liquid (B) passes only through dialyzer DB and not through
adsorbents.
The conventional dialyzer (DB) is connected to a conventional
dialysate liquid which may be coming from a conventional dialysis
machine or pumped by other perfusor or roller pumps from bags.
DETAILED DESCRIPTION OF THE INVENTION
A. Method for separating protein-bound substances from
protein-containing liquids
The present invention provides a practical and effective method for
the removal of undesired or potentially harmful protein-bound
substances and/or lipophilic substances from protein-containing
liquids such as plasma and blood.
The basic procedure is similiar to conventional high flux dialysis
with modifications according to the present invention as described
below.
1. The dialysate liquid (B)
The dialysate liquid (B) contains a protein serving as an acceptor
for the protein-bound substances (PBS) to be removed from the
liquid (A). The acceptor protein should have a sufficient affinity
towards the substances which are bound to the protein in liquid
(A). A preferred acceptor protein is human serum albumin. The
concentration of the acceptor protein is from about 1 to about 50
g/100 ml, preferably from about 6 to about 40 g/100 ml, more
preferably from about 8 to about 30 g/100 ml and most preferably
from about 8 to about 20 g/100 ml.
The dialysate liquid (B) contains furthermore salts like NaCl, KCl,
MgCl.sub.2, CaCl.sub.2, sodium lactate and glucose monohydrate in
amounts depending on the electrolyte composition in the blood of
the specific patient. For example, in the dialysis of a patient
suffering hypopotassemia a higher concentration of potassium ions
is required.
Preferred ion concentrations in a dialysate liquid (B) that is
bicarbonate buffered are for sodium from about 130 to about 145
mmol/1000 ml, for calcium from about 1.0 to about 2.5 mmol/1000 ml,
for potassium from about 2.0 to about 4.0 mmol/1000 ml, for
magnesium from about 0.2 to about 0.8 mmol/1000 ml, for chloride
from about 100 to about 110 mmol/1000 ml, for bicarbonate from
about 30 to about 40 mmol/1000 ml, for acetate from about 2 to
about 10 mmol/1000 ml, for human serum albumin from about 1 to
about 50 g/100 ml, preferably from about 6 to about 40 g/100 ml,
more preferably from about 8 to about 30 g/100 ml, and most
preferably from about 8 to about 20 g/100 ml.
More preferred ion concentrations in a dialysate liquid (B) that is
bicarbonate buffered are for sodium from about 135 to about 140
mmol/1000 ml, for calcium from about 1.5 to about 2.0 mmol/1000 ml,
for potassium from about 3.0 to about 3.5 mmol/1000 ml, for
magnesium from about 0.4 to about 0.6 mmol/1000 ml, for chloride
from about 104 to about 108 mmol/1000 ml, for bicarbonate from
about 34 to about 38 mmol/1000 ml, for acetate from about 4 to
about 8 mmol/1000 ml, for human serum albumin from about 1 to about
50 g/100 ml, preferably from about 6 to about 40 g/100 ml, more
preferably from about 8 to about 30 g/100 ml, and most preferably
from about 8 to about 20 g/100 ml.
Preferred ion concentrations in a dialysate liquid (B) that is
acetate buffered are for sodium from about 130 to about 145
mmol/1000 ml, for calcium from about 1.0 to about 2.5 mmol/1000 ml,
for potassium from about 2.0 to about 4.0 mmol/1000 ml, for
magnesium from about 0.2 to about 0.8 mmol/1000 ml, for chloride
from about 100 to about 110 mmol/1000 ml, for acetate from about 30
to about 40 mmol/1000 ml, for human serum albumin from about 1 to
about 50 g/100 ml, preferably from about 6 to about 40 g/100 ml,
more preferably from about 8 to about 30 g/100 ml, and most
preferably from about 8 to about 20 g/100 ml.
More preferred ion concentrations in a dialysate liquid (B) that is
acetate buffered are for sodium from about 135 to about 140
mmol/1000 ml, for calcium from about 1.5 to about 2.0 mmol/1000 ml,
for potassium from about 3.0 to about 3.5 mmol/1000 ml, for
magnesium from about 0.4 to about 0.6 mmol/1000 ml, for chloride
from about 104 to about 108 mmol/1000 ml, for acetate from about 33
to about 38 mmol/1000 ml, for human serum albumin from about 1 to
about 50 g/100 ml, preferably from about 6 to about 40 g/100 ml,
more preferably from about 8 to about 30 g/100 ml, and most
preferably from about 8 to about 20 g/100 ml.
An example for a dialysate liquid (B) comprises from about 10 to
about 20% by weight human serum albumin, about 6.1 g NaCl, about
4.0 g sodium lactate, about 0.15 g KCl, about 0.31 g CaCl.sub.2
.times.2 H.sub.2 O, 0.15 g MgCl.sub.2 .times.6 H.sub.2 O, and 1.65
g glucose monohydrate per liter of dialysate liquid (B).
2. The membrane
The membrane of the present invention preferably comprises two
functionally different parts (regions). One part has the actual
separating membrane function permitting the PBS and the
water-soluble substances to pass through under the conditions of
the process of the present invention and excluding the protein(s)
which had bound the PBS in liquid (A) and the acceptor protein of
liquid (B), and the other part has a port- and adsorption function.
Preferably, the membrane is coated with a protein having an
acceptor function for the PBS. In a preferred embodiment the
membrane of the present invention comprises a thin layer of a
tunnel-like structure facing the liquid (A) side, the tunnels
having a length less than about 10 .mu.m, preferably less than
about 5 .mu.m, more preferably less than about 0.1 .mu.m and most
preferably between about 0.01 and about 0.1 .mu.m. The tunnels have
a diameter sufficiently small to exclude the protein in liquid (A),
preferably to permit the passage of molecules having a molecular
weight from about 20,000 daltons to about 66,000 daltons, more
preferably from about 50,000 to about 66,000 daltons through the
tunnels. Preferably the sieve coefficient of the membrane with
respect to the protein in liquid (A) is less than 0.1, more
preferably less than 0.01. Furthermore, the membrane preferably
comprises a port- and adsorption-structure on the dialysate liquid
(B) side. This part has to provide a structure sufficiently open to
permit the acceptor protein in the dialysate liquid (B) to enter
the port- and adsorption layer to accept the PBS coming from the
liquid (A) side of the membrane. Moreover the internal surface of
this part acts as an adsorber for the PBS via the acceptor protein
that is adsorbed by the coating procedure described in the
following or by other structures suitable for binding the PBS. This
adsorption can either be stable over time or reversible. Preferably
the membrane is coated on at least one side with a thin film of a
protein having an acceptor function for the protein-bound
substances. A commercial dialyzer comprising a membrane of the
present invention may contain on the liquid (B) side a solution of
the acceptor protein.
The membrane of the present invention may have the macroscopic form
of a flat film, a thin-walled but large diameter tube, or
preferably fine hollow fibers.
The matrix material for the membrane may be made from various
materials, including ceramics, graphite, metals, metal oxides, and
polymers, as long as they have an affinity towards the protein on
the liquid (A) and the dialysate liquid (B). The methods used most
widely today are sintering of powders, stretching of films,
irradiation and etching of films and phase inversion techniques.
The preferred materials for the membranes of the present invention
are organic polymers selected from the group consisting of
polysulfones, polyamides, polycarbonates, polyesters, acrylonitrile
polymers, vinyl alcohol polymers, acrylate polymers, methacrylate
polymers, and cellulose acetate polymers.
The preferred polymer membranes used in the present invention are
highly permeable asymmetric polysulfone membranes hydrophilized
with e.g. polyvinylpyrrolidone, e.g. HF 80 of Fresenius AG.
Such membranes and membrane modules, dialysis cardriges, artificial
kidney membrane systems are commercially available for instance
from Fresenius AG, Polyflux from GAMBRO AB, CT190G from Baxter
Inc.
First part: The layer or structure of the membrane facing the
liquid (A) side has to provide the actual membrane permitting a
selective transfer of protein-bound substances and water-soluble
substances, i.e. low-molecular substances and "middle molecules"
from the liquid (A) side to the dialyzing solution (liquid (B)
side). Thus, an effective net transport of undesired substances
occurs from the liquid (A) side to the dialysate liquid (B) side
following the concentration gradient for the undesired substances
decreasing from the liquid (A) side towards the dialysate liquid
(B) side. Three conditions have to be met for the actual
membrane:
1. The tunnels have to be sufficiently short, preferably less than
about 5 .mu.m, more preferably less than about 1 .mu.m, and most
preferably less than about 0.1 .mu.m.
2. The tunnel diameter has to be sufficiently large to permit
passage of the undesired molecules and sufficiently small to
inhibit passage of the desired molecules contained in liquid (A)
towards liquid (B) and of the acceptor protein from liquid (B) to
liquid (A). In case of plasma or blood as liquid (A) the exclusion
limit is preferably about 66,000 daltons. Preferably the sieve
coefficient of the membrane with respect to the protein in liquid
(A) is less than 0.1, more preferably less than 0.01.
3. The chemical, physical etc. structure of the layer or structure
of the actual membrane facing the liquid (A) side is such that
passage of the undesired substances is permitted, e.g. by
hydrophobic and hydrophilic microdomains.
Second part: The layer or structure of the membrane facing the
liquid (B) side has to provide a more open membrane structure
normally in a sponge- or finger-like fashion. This part provides an
important port- and adsorption-function within this part of the
membrane:
1. Due to the open-spaced structure of this part of the membrane
the acceptor protein coming from the dialysate liquid (B) side can
approach the dialysate side ostium of the structure facing the
liquid (A) side described above and accept undesired substances,
such as protein-bound substances passing through the tunnel-like
structure from the liquid (A) side.
2. Due to the large total surface area present in this structure it
adsorbs remarkable amounts of the protein-bound substances (PBS)
via attached molecules that function as a kind of spacer in this
mediate membrane adsorption or the PBS are directly membrane bound
if the membrane has a capacity to adsorb the PBS due to its own
structure. This adsorption can either be reversible or irreversible
but preferably it is reversible.
3. Due to the open structure towards the dialysate liquid (B) side
of the membrane a dialysate movement that might be directed
perpendicular or in parallel to the outer membrane surface or in a
different fashion can transport acceptor protein molecules both
into the port layer and out of the port layer. Preferably the
movement and the transport perpendicular to the outer membrane
surface is provided by an alternating influx and outflux movement
of liquid (B) that moves into the port membrane and back out into
the liquid (B) stream. This influx and outflux can be provided by a
pulse-like pressure profile obtained by the use of roller pumps or
a change in transmembranal pressure changing along the membrane
from being directed towards the liquid (B) first (positive TMP) and
to the liquid (A) at last (negative TMP); TMP=transmembranal
pressure.
Thus, the dialysis membrane of the present invention preferably is
functionally divided into a tunnel-like part and a finger- or
sponge-like port/adsorption part. Both of them have to fullfill
certain prerequisites to render the method of the present invention
possible. The ideal tunnel-like part would be one with a length
next to zero (0.01 to 0.1 .mu.m), a diameter next to the size of
the desired protein to be purified and kept in the retentate, e.g.
the diameter of albumin. In other words, the tunnel-like part
should have a diameter sufficiently small to retain valuable and
desired substances of the liquid (A) in the retentate and to permit
protein-bound substances and other undesired substances contained
in liquid (A) to pass to the dialysate liquid (B) side.
The ideal port/adsorption part of the dialysis membrane of the
present invention has a very open structure to enable the acceptor
protein to approach and leave the area next to the dialysate side
of the tunnel. It has a large inner surface which adsorbs the PBS
directly or via the attached acceptor protein. The total diameter
of this part should again be as small as possible to render the
exchange into the dialysate stream more effective. The latter two
points can be brought to their extremes almost excluding the other
one according to whether more adsorption or more transit through
the port/adsorption part of the membrane is desired.
Conventional dialysis membranes for purifying e.g. plasma or blood
can be classified by functional or structural criteria. Functional
criteria are high flux, low flux or highly permeable, whereas
structural criteria are e.g. flat, hollow fiber, symmetric or
asymmetric. The group of tunnel-like membranes (TM) useful for the
present invention is not sufficiently described by these terms
because
a) TM are high flux and highly permeable membranes but not every
high flux membrane named "highly permeable" is a TM (e.g. AN69 from
HOSPAL);
b) TM can be asymmetric but not every asymmetric membrane is a TM
(e.g. F8 from FRESENIUS AG);
c) TM can be asymmetric and highly permeable but not every
asymmetric and highly permeable membrane is a TM (PMMA from
Toray)
d) TM can be symmetric but not every symmetric membrane is a TM
(e.g. Cuprophan from AKZO).
Therefore the term tunnel-like membrane represents a new quality of
structural and functional features of dialysis membranes useful for
the present invention.
4. Pretreatment and conditions of the membrane
The membrane of the present invention preferably is pretreated as
follows. The membrane is impregnated on at least one side,
preferably both from the liquid (A) side and from the liquid (B)
side with a solution of the acceptor protein. A preferred solution
for the impregnating step is a 0.9% NaCl solution, containing the
acceptor protein, preferably human serum albumin, in a
concentration from about 1 to about 50 g/100 ml, preferably from
about 6 to about 40 g/100 ml, more preferably from about 8 to about
30 g/100 ml, and most preferably from about 8 to about 20 g/100 ml.
The impregnating solution is passed along the liquid (A) side and
the liquid (B) side of the membrane for a time sufficient to permit
penetration and adsorption of the acceptor protein on the two parts
of the membrane, in general from about 1 to about 120 min,
preferably from about 10 to about 60 min, at a temperature from
about 15.degree. to about 40.degree. C., preferably from about
18.degree. to about 37.degree. C., the pH value being from about 5
to about 9, preferably about 7. The pretreatment can be carried out
immediately prior to use of the membrane, but the pretreated
membrane may also be stored under sterile conditions at a
temperature up to 24.degree. C. for up to two years if the acceptor
protein is human serum albumin. Preferably the impregnating
solution is pumped by roller pumps exhibiting a "pulse like
pressure profile" during the coating procedure, e.g. by two roller
pumps, one on the dialysate side compartment and one on the blood
side compartment of the dialyzer. Preferably there is a phase delay
between the pressure profiles of the two pumps thus to ensure an
effective in- and outflow of the solution on both sides of the
membrane.
5. Procedure for the separation of protein-bound substances (PBS)
from a protein containing liquid (A)
The procedure for the separation of PBS from a protein-containing
liquid (A) preferably is carried out as follows: Liquid (A) to be
purified is passed along the liquid (A) side of the dialysis
membrane of the present invention with a flow rate from about 50 to
about 300 ml/min, preferably from about 100 to about 200 ml/min per
sqm of the dialysis membrane. The dialysate liquid (B) is passed
along the dialysate side (B) of the membrane with a flow rate from
about 50 to about 1000 ml/min, preferably from about 100 to about
500 ml/min per sqm of the dialysis membrane. The flow rates of the
liquid (A) and thus liquid (B) are preferably in the same order of
magnitude. The ratio of the flow rate of liquid (A) to liquid (B)
is from about 1:0.1 to about 1:10, preferably from about 1:1 to
about 1:5. The retentate is the purified protein-containing liquid
(A) from which protein-bound substances and other undesired
substances are removed.
In a preferred embodiment of the process of the present invention
the first dialysis step of the liquid (A) is combined with two
steps of aftertreatment of the dialysate liquid (B) obtained.
First the dialysate liquid (B) obtained is passed through a second
conventional dialyzer which is connected to a conventional dialysis
machine. Dialysis is carried out against an aqueous standard
dialysate liquid. By this dialysis water-soluble substances can be
exchanged between the dialysate liquid (B) and a standard dialysate
liquid.
Water-soluble toxins, urea and/or creatinine are removed from the
dialysate liquid (B), and electrolytes, glucose and the pH value
can be balanced in the dialysate liquid (B) which is the retentate.
The dialysate liquid (B) is thereafter passed through a
charcoal-adsorbent, e.g. Adsorba 300 C from GAMBRO AB or N350 from
ASAHI, and then through an anion exchange column, e.g. BR350 from
ASAHI, to remove the protein-bound substances from the acceptor
protein in the dialysate liquid (B). The purified acceptor
protein-containing dialysate liquid (B) is then returned to the
liquid (B) side of the membrane of the present invention.
This procedure has been tested experimentally for the separation of
albumin-bound drugs and toxins in a protein-containing liquid and
led to a significant reduction of these compounds in the
liquid.
Other possible simplified embodiments of the procedure of the
present invention comprise the following modifications. The
dialysate liquid (B) coming from the dialyzer may be passed through
another dialyzer but not through any adsorbent. The dialysate
liquid (B) coming from the dialyzer may be passed through one or
two adsorbents but not through another dialyzer. The dialysate
liquid (B) coming from the dialyzer may be pumped directly back
into the inlet of the dialysate compartment of the dialyzer (e.g.
by a roller pump) thus realizing a sufficient movement of the
dialysate liquid (B) and sufficient removal of ABT. A further
simple modfication would be a dialyzer with a dialysate compartment
filled with the dialysate liquid (B) comprising human serum albumin
in a concentration of from about 1 to about 50 g/dl, preferably
from about 6 to about 40 g/dl, more preferably between 8 and 30
g/dl, and most preferably from about 8 to about 20 g/dl that is
closed at the dialysate inlet and outlet. The whole dialyzer may be
moved, e.g. by shaking or rolling.
The benefit of the method of the present invention is that a
biological protein-containing liquid such as blood or plasma
contaminated with potentially harmful or undesired protein-bound
substances and possibly undesired water-soluble substances can be
selectively purified by the method of this invention so that the
biological liquid contains the protein-bound substance and the
other undesired substances in a lower concentration than before and
does not exhibit the potentially harmful or unwanted effects which
it had prior to the dialysis treatment.
Another benefit of the method of the present invention is that the
chemical and physical homeostasis of the biological liquid remains
almost unchanged, i.e. the method and the dialysis membrane used
for it exert good biocompatibilty.
The method of the present invention furthermore has the advantage
that it is simple, practical and suitable for treating large
volumes of biological liquids, e.g. blood in an extracorporeal
circuit repeatingly for hours and for different conditions with
commercially available dialysis machines.
A disposable set for the separation of protein-bound substances and
water-soluble substances from plasma or blood containing said
substances can comprise a membrane as described before, a second
conventional dialyzer for hemodialysis, a conventional charcoal
adsorber unit for hemoperfusion, and a conventional ion exchange
resin unit for hemoperfusion interconnected by tubing and a unit of
a human serum albumin containing dialysate liquid.
A disposable set for the separation of protein-bound substances and
water-soluble substances from plasma or blood containing said
substances may as well comprise a dialyzer having a membrane as
described before and being filled on the dialysate liquid (B) side
with a human serum albumin containing liquid, a second conventional
dialyzer for hemodialysis, a conventional charcoal adsorber unit
for hemoperfusion, and a conventional ion exchange resin unit for
hemoperfusion interconnected by tubing and a unit of a human serum
albumin containing dialysate liquid.
B. Evaluation of removal of PBS and water soluble substances
All compounds were determined immediately after the experiment in
order to avoid degradation especially of the light instable
bilirubin.
Bromosulfophthalein: Spectrophotometrically according to the method
of BECH
Bilirubin: a) Spectrophotometrically according to the method of
JENDRASSIK; b) KODAK EKTACHEM dry chemistry. Both methods showed
good correlation in the range of concentrations used.
Phenols: a) Spectrophotometrically according to the method of
BECHER; b) HPLC. Again both methods showed good correlation in the
range of concentrations used.
Free fatty acids according to the method of LAURELL and TIBBLING
(i.e. fatty acids that have not formed esters)
Digoxin: ABBOTT analyzer/dry chemistry
Digitoxin: ABBOTT analyzer/dry chemistry
creatinine, urea, uric acid, transaminases, blood cell counts:
KODAK EKTACHEM dry chemistry.
C. The in vitro model
A conventional dialysis machine with two closed loop compartments
(a plasma compartment and a compartment for dialysis solution) is
used in order to permit the measurement not only of the decreasing
concentrations in the biological liquid but also the increasing
concentrations in the dialysate which is the essential criterion to
distinguish a membrane transport process from an adsorption to the
membrane. Two glas bottles about 25 cm high and about 10 cm
diameter with a volume of 500 ml each are used, one as container
for the biological liquid containing plasma the other as container
for the dialysate solution. Both bottles are have connectors for
two tubes, one for the inflow and one for the outflow of plasma or
dialysate respectively. The distance between the inflow and the
outflow should not be smaller than 10 cm in order to avoid "shunt
circulation". The two connectors of each bottle are connected with
two flexible plastic tubes (about 100 cm and about 6 mm diameter)
each with a silicone segment permitting the use of a conventional
roller pump for dialysis. The other end of the two tubes from the
bottle containing the plasma are connected with the connectors of
the blood side of a dialysator in the following manner: the outflow
of the bottle is connected with the inflow of the dialysator, the
outflow of the dialysator is connected with the inflow of the
bottle. The other end of the two tubes from the bottle containing
the dialysate are connected with the connectors of the dialysate
side of the dialysator in the following manner: The outflow of the
bottle is connected with the dialysate inflow of the dialysator and
the dialysate outflow of the dialysator is connected with the
inflow of the bottle. The plasma inflow of the dialysator is placed
next to the dialysate outflow in order to design the dialysate flow
through the dialysator countercurrently to the plasma flow as it is
known from clinical hemodialysis. The silicone segments of the
inflow/outflow tubes of the plasma bottle are used to pump the
plasma by a simultane roller pump. The silicone segments of the
inflow/outflow tubes of the dialysate bottle are used to pump the
dialysate by a simultane roller pump also. In this way the
inflow/outflow volumes of both compartments are balanced and
transmembranal water losses which could influence the results are
avoided.
Preparation of the plasma used for in vitro experiments
a) For evaluation of the method of the present invention using a
polysulfone dialysis membrane
Human heparinized plasma was taken from young male donors and
enriched with model substances with a high protein binding
rate:
--caprylic acid (750 mg/1000 ml)
--phenol (530 mg/1000 ml)
--unconjugated bilirubin (11 mg/100 ml)
--sulfobromophthalein (230 mg/1000 ml)
110 mg unconjugated bilirubin, 230 mg sulfobromophthalein, 750 mg
caprylic acid, and 530 mg phenol were dissolved in 50 ml of a 0.1M
NaOH solution. Thereafter 2.5 g human albumin (fatty acid free,
SIGMA) were dissolved in the solution. Thereafter the pH value was
adjusted to 7.4 by addition of a 30 percent acetic acid solution.
Thereafter this toxin cocktail was mixed with 950 ml heparinized
plasma. 500 ml of this plasma solution were filled into the plasma
bottle.
Composition of the dialysate: 100 ml of a 20 percent (20 g/100 ml)
human albumin solution were mixed with 400 ml of a commercial CVVH
dialysis solution. 500 ml of this albumin containing solution were
filled into the dialysate bottle.
b) For evaluation of other dialysis membranes/hemofilters
Human heparinized plasma was taken from young male donors and
enriched with model substances with a high protein-binding
rate:
--unconjugated bilirubin (11 mg/100 ml)
--sulfobromophtaleine (230 mg/1000 ml)
as well as with model substances wich are known as markers of
uremia and effectivity of dialysis:
--creatinine (6 mg/100 ml)
--urea (100 mg/100 ml)
--uric acid (17 mg/100 ml)
110 mg unconjugated bilirubin, 230 mg sulfobromophthalein, 60 mg
creatinine, 1000 mg urea, and 170 mg uric acid were dissolved in 50
ml of a 0.1M NaOH solution. Thereafter 2.5 g human albumin (fatty
acid free, SIGMA) were dissolved in the solution. Thereafter the pH
value was adjusted to 7.4 by addition of a 30 percent acetic acid
solution at room temperature. Thereafter this toxin cocktail was
mixed with 950 ml heparinized human plasma. 500 ml of this plasma
solution were filled into the plasma bottle.
After filling the bottles they were closed hermetically and the
pump segments (inflow and outflow) installed to the simultane
roller pumps in a manner permitting recirculation of each (plasma
and dialysate) circle counter-currently. The flow rates were
adjusted at 100 ml/min. The temperature was adjusted to 37.degree.
C.
The following dialysis membranes/hemofilters were used:
a) For the evaluation of the principle of the method of the present
invention
HF 80, FRESENIUS AG, Germany
b) For the evaluation of other dialysis membranes
Diafilter 30, AMICON, U.S.A.
FH 88, GAMBRO AB, Sweden
LUNDIA PRO 5, GAMBRO AB, Sweden
CT 110 G, BAXTER, U.S.A.
SPAN, ORGANON TECHNIKA, Netherlands
KF 201 EVAL N 13, KAWASUMI, Japan
B1-2.1U, TORAY Medical Co., Japan
FILTRAL 16, HOSPAL, France
Dialysis membranes, technical information from the firms
*The highly permeable FRESENIUS AG polysulfone membrane is a
asymmetric membrane with a 0.5 to 1 .mu.m skin layer determining
the cut off (about 40,000 daltons). The skin layer is supported by
a macroporous support layer of about 40 .mu.m thickness for the
High flux dialysis membrane and of about 35 .mu.m thickness for the
hemofiltration membrane. The hydrophobic surface structure of the
polysulfone membrane is hydrophilized by polyvinylpyrrolidone.
The low permeable FRESENIUS AG polysulfone hollow fiber membrane is
a asymmetric membrane with a 0.5 to 1 .mu.m skin layer determining
the cut off (ca 5000 daltons) which is supported by a macroporous
support layer of about 40 .mu.m thickness. The hydrophobic surface
structure of the polysulfone membrane is hydrophilized by
polyvinylpyrrolidone. These fibers are present in the following
Fresenius dialyzers: F3, F4, F5, F6, F7, F8.
*The HOSPAL PAN membrane (AN 69) is a symmetric membrane based on
polyacrylonitrile with a high permeability and a membrane thickness
of 50 .mu.m. This membrane is present in Filtral 8-20 as a hollow
fiber and in Biospal.
*The ORGANON TECHNIKA SPAN membrane is an asymmetric membrane based
on polyacrylonitrile.
*The DIAFILTER is an asymmetric hollow fiber polysulfone hemofilter
membrane with a skin layer determining the cut off supported by a
macroporous support layer. This hemofiltration membrane is present
in the following AMICON filters: Minifilter, Minifilter plus,
Diafilter 10, 20, 30, and 50.
*The Lundia pro 5 membrane is a flat polycarbonate dialysis
membrane.
*The GAMBRO AB polyamide hollow fiber membranes are asymmetric
membranes sold in three modifications:
Polyamide for high flux dialysis and hemodiafiltration with a wall
thickness of 50 .mu.m, designed as a 3-layer and of high
hydrophobicity. The internal diameter of the hollow fiber is 220
.mu.m. These hollow fibers are present in Polyflux 130 and Polyflux
150 (Trademark names).
Polyamide for hemofiltration with a wall thickness of 60 .mu.m,
designed as a 3-layer and of a high hydrophilicity. The internal
diameter of the hollow fiber is 215 .mu.m. These hollow fibers are
present in FH 22, FH 77, FH 88 and with a wall thickness of 50
.mu.m in FH 66 (Trademark names).
Polyamide for water ultrafiltration (for the separation of
pyrogens) with a wall thickness of 60 .mu.m designed as a "finger
structure" of lower hydrophilicity. The internal diameter of the
hollow fiber is 215 .mu.m. These hollow fibers are present in U
2000 and U 7000 (Trademark names).
*The KF 201 EVAL N13 is a ethylene-vinyl alcohol copolymer hollow
fiber membrane with a wall thickness of 32 .mu.m. This membrane is
also present in KF 101 EVAL N16.
*The TORAY PMMA hollow fiber membrane is an asymmetric membrane on
the basis of polymethylmethacrylate with a wall thickness of 30
.mu.m in B1-1.6U and B1-2.1U or of 20 .mu.m in B2-0.5 up to B2-2.0
and B1-0.6H up to B1-1.6H.
*The BAXTER CT 100 G is a cellulose triacetate hollow fiber
membrane. It has been described as a hybrid cellulose material with
a symmetric pore size distribution and a wall thickness of 15
.mu.m. This type of hollow fibers is also present in BAXTER CT 190
G.
Each dialyzer was impregnated before its use by preperfusion with a
human serum albumin solution (5 g/100 ml) on the blood side as well
as on the dialysate side with a flow rate of 50 ml/min for a period
of 20 min.
Abbrevations in the tables:
______________________________________ Alb. Albumin Un. Bil.
unconjugated Bilirubin c. Bil. conjugated Bilirubin SBP
Sulphobromophtalein Ph Phenol FFA Free fatty acid, in this case
caprylic acid DIG Digitoxin Crea Creatinine Ur. acid Uric acid
______________________________________
D. In vitro Results
a) Evaluation of the Principle
______________________________________ Tab. 1 Removal of PBS from
plasma (liquid A) in vitro (HF 80, Fresenius AG), plasma
concentrations (in liquid A): Time Alb. Un. Bil. SBP Ph FFA (min)
(g/dl) (mg/dl) (mg/l) (mg/l) (mg/l)
______________________________________ 0 3.5 11.70 230 529 749 5
3.4 9.36 186 370 562 10 3.4 8.42 151 291 502 30 3.4 7.72 133 190
434 60 3.4 7.02 124 137 419 90 3.3 5.85 117 127 412
______________________________________
In order to demonstrate the importance of the albumin in the
dialysate liquid (B) a comparative test was performed under the
same conditions as in Tab. 1 but omitting the albumin in the
dialysate liquid (B). The results are shown in Comparative
Tab.1.
______________________________________ Comparative Tab. 1. Removal
of PBS from plasma (liquid A) in vitro (HF 80, Fresenius AG),
plasma concentrations (in liquid A) using a conventional dialysate
liquid without albumin: Time Alb. Un. Bil. SBP Ph FFA (min) (g/dl)
(mg/dl) (mg/l) (mg/l) (mg/l) ______________________________________
0 3.5 11.70 230 529 749 5 3.4 11.5 227 420 730 10 3.4 11.42 221 371
723 30 3.4 11.44 218 290 719 60 3.4 11.39 211 270 718 90 3.3 11.40
215 268 724 ______________________________________
______________________________________ Tab. 2. Removal of PBS from
plasma (Liquid A) (HF 80, Fresenius AG), dialysate concentrations
(in liquid B) Time Alb. Un. Bil. SBP Ph FFA (min) (g/dl) (mg/dl)
(mg/l) (mg/l) (mg/l) ______________________________________ 0 4.0
0.00 0 0 0 5 3.0 0.9 21 52 149 10 3.1 1.5 48 79 187 30 2.9 1.7 67
100 202 60 2.9 2.1 103 111 217 90 2.8 2.3 119 116 224
______________________________________
A comparative test was performed under the same conditions as in
Tab. 2 but omitting the albumin in the dialysate liquid (B). The
results are shown in Comparative Tab.2.
______________________________________ Comparative Tab. 2. Removal
of PBS from plasma (Liquid A) (HF 80, Fresenius AG), dialysate
concentrations (in liquid B) using a conventional dialysate liquid
without albumin: Time Alb. Un. Bil. SBP Ph FFA (min) (g/dl) (mg/dl)
(mg/l) (mg/l) (mg/l) ______________________________________ 0 0.0
0.0 0 0 0 5 <0.2 0.0 0 28 0 10 <0.2 0.0 0 79 0 30 <0.2 0.0
0 121 0 60 <0.2 0.0 0 173 0 90 <0.2 0.0 0 181 0
______________________________________
b) From Different Types of Dialyzers/Membranes
______________________________________ Tab. 3. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (Amicon
Diafilter), plasma concentrations (liquid A). time Alb SBP Un. Bil.
c. Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) (mg/dl) ______________________________________ 0
3.5 230 11.7 0.6 6.8 120 16.8 30 3.4 86 4.5 0.47 3.2 37 9.8 60 3.3
56 3.9 0.40 2.9 37 8.1 ______________________________________
______________________________________ Tab. 4. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (Amicon
Diafilter), dialysate concentrations (liquid B). time Alb SBP Un.
Bil. c. Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) (mg/dl) ______________________________________ 0
4.0 0 0.00 0.00 0.0 0 0.0 30 3.3 9 0.46 0.14 3.6 47 10.3 60 3.3 44
0.91 0.36 3.5 47 10.3 ______________________________________
______________________________________ Tab. 5. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (FH 88 from
GAMBRO). plasma concentrations (liquid A). time Alb SBP Un. Bil. c.
Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) ______________________________________ 0 3.5 230
11.7 0.6 6.8 120 16.8 30 3.2 47 7.3 0.13 2.8 19 3.0 60 3.1 32 6.3
0.13 2.7 12 3.0 ______________________________________
______________________________________ Tab. 6. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (FH 88 from
GAMBRO), Dialysate concentrations (liquid B). time Alb SBP Un. Bil.
c. Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) (mg/dl) ______________________________________ 0
4.0 0 0.00 0.00 0.0 0 0.0 30 3.5 33 1.08 0.07 3.5 30 6.9 60 3.3 49
1.68 0.12 3.4 28 6.9 ______________________________________
______________________________________ Tab. 7. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (CT 100 G from
BAXTER), plasma concentrations (liquid A). time Alb SBP Un. Bil. c.
Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) ______________________________________ 0 3.4 232
11.7 0.6 6.8 120 16.8 30 3.3 114 8.8 0.5 3.0 29 8.2 60 3.2 84 7.6
0.45 3.0 38 8.3 ______________________________________
______________________________________ Tab. 8. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (CT 110 G from
BAXTER), Dialysate concentrations (liquid B). time Alb SBP Un. Bil.
c. Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) (mg/dl) ______________________________________ 0
4.0 0 0.00 0.00 0.0 0 0.0 30 3.5 59 0.8 0.02 3.0 39 8.1 60 3.3 84
1.1 0.02 3.0 38 8.1 ______________________________________
______________________________________ Tab. 9. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (SPAN from
Organon Technika), Plasma concentrations liquid A). time Alb SBP
Un. Bil. c. Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl)
(mg/dl) (mg/dl) (mg/dl) (mg/dl)
______________________________________ 0 3.5 240 11.7 0.6 6.8 120
16.8 30 3.2 167 9.2 0.58 27 38 6 60 3.2 148 8.6 0.58 2.5 38 6
______________________________________
______________________________________ Tab. 10. In vitro removal of
PBS and hydrophilic toxins from plasma (liquid A) (span von Organon
Technika), Dialysate concentrations (liquid B). time Alb SBP Un.
Bil. c. Bil. Crea Urea Ur. acid (min) (g/dl) (mg/l) (mg/dl) (mg/dl)
(mg/dl) (mg/dl) (mg/dl) ______________________________________ 0
4.0 0 0.00 0.00 0.0 0 0.0 30 3.6 6 0.19 0.17 2.6 38 7.0 60 3.6 18
0.19 0.18 2.6 38 7.0 ______________________________________
c) Influence of Different Albumin Concentrations in the Dialysate
Liquid (B)
Phenol-enriched plasma: Human heparinized plasma was taken from
young male donors and enriched with phenol. This was performed by
dissolving 530 mg phenol in 50 ml of a 0.1M NaOH solution.
Thereafter 2.5 g human albumin (fatty acid free, SIGMA) were added.
Thereafter the pH value was adjusted to 7.4 with a 30 percentage
acetate solution. Finally this toxin cocktail was mixed with 950 ml
pooled plasma from healthy young male donors. 500 ml of this plasma
solution were filled into the plasma bottle on the plasma side of a
dialyzer.
Dialysate liquid (B):200 ml of a 20 percent (20 g/100 ml) human
albumin solution were mixed with 300 ml of a commercial CVVH
dialysis solution. 500 ml of this solution (i.e. 80 g/l albumin)
were filled into the dialysate bottle on the dialysate side of a
dialyzer.
After filling the bottles they were closed hermetically and the
pump segments (inflow and outflow) installed to the simultane
roller pumps in a manner enabeling recirculation of each (plasma
and dialysate) circle counter currently. The flow rates were
adjusted to 100 ml/min. The temperature was adjusted to 37.degree.
C.
______________________________________ Tab. 11. Removal of phenol
from plasma (liquid A) in vitro (HF 80, FRESENIUS AG) with enhanced
albumin concentration in the dialysate (8 g/dl), plasma and
dialysate concentrations time Phenol concentration (mg/l) (min)
liquid A (plasma) liquid B (dialysate)
______________________________________ 0.0 529 0 2.5 158 148 5.0
132 151 10.0 116 148 20.0 105 147
______________________________________
E. In vivo System
A commercial dialysis machine (A 2008, Fresenius) was chosen as
hardware equipment for the in vivo system. Every single piece was
commercially available and had safety approval from the German
Federal Health Authority, Berlin, for usage in a extracorporeal
therapeutic blood treatment.
The blood pump of the A 2008 was used to supply continuous blood
flow of 160 ml/min through an asymmetric polysulfone hollow fiber
dialyzer (1.8 sqm, HF 80, Fresenius), that was on both sides
albumin impregnated as described above. The liquid (B) dialysate
compartment was a closed loop system which contained 1000 ml of
Ringers lactate solution with albumin in a concentration of 5 g/100
ml (i.e. a concentration used for intravenuous infusions or liquid
replacement in plasma exchange). The flow rate of this albumin
containing liquid (B) dialysate of 120 ml/min was realized by the
second pump of the machine (normally used for substitution
liquids). Moreover, the in vitro-system was modified by introducing
a three-step regeneration of the albumin-containing liquid (B)
dialysate, thus increasing the PBS-separation capacity and adding
the possibility of effective hemofiltration and separation of water
soluble toxins.
The liquid (B) dialysate loop contained in this order:
1. An additional conventional large pore dialyzer (1.3 sqm
polysulfon hollow fiber dialyzer, HF 60, FRESENIUS AG) which was
connected to the normal dialysate compartment of the A 2008. While
the albumin-containing closed-loop liquid (B) dialysate was passed
through the blood compartment of the dialyzer, a standard
bicarbonate dialysate (potassium ions 4 mmol/liter) was employed on
the other side. The aim of this first step was to remove
water-soluble factors of hepatic coma (ammonia, imbalanced amino
acids, conjugated bilirubin etc.) to support electrolyte-, glucose-
and pH-regulation and to support or replace kidney function in
hepatorenal syndrome or other types of kidney failure.
2. A commercial column of activated charcoal (Adsorba 300C, GAMBRO
AB). The aim of this second step was to remove a first group of PBS
(e.g. aromatics, fatty acids) from the albumin in order to
facilitate the reuse of the albumin solution in the closed-loop
system.
3. A commercial anion exchange resin (Plasorba BR-350, Asahi
Medical). The aim of this third step was the separation of
unconjugated bilirubin and accumulated bile acids from the albumin
solution.
After this regeneration the albumin solution was recirculated into
the liquid (B) dialysate compartment of the HF 80 dialyzer for
further blood purification.
F. In vivo Results
*Patient 1
The patient was a 30 year old Caucasian woman with an acute
decompensation of a chronic liver failure after a six year history
of alcoholism. The patient was treated for 10 days conservatively
without success. At entry to the treatment the patient was in grade
IV hepatic encephalopathy (i.e. deep coma, patient not reacting to
painful stimuli). Hypotension, hypoglycemia and alcalosis were
present.
Biochemistry: thromboplastine time (Quick) 19%, activated
coagulation time was higher than 200 seconds, antithrombine III was
18%, platelet count was 73.times.10.sup.9 /liter (with an
decreasing tendency). The total bilirubin was 615 .mu.mol/liter,
unconjugated bilirubin 51 .mu.mol/liter, cholinesterase 16
.mu.mol/liter, ammonia 96 .mu.mol/liter. The amount of
aminotransferases was five to tenfold increased, the blood pH was
7.5. The patient underwent three treatment procedures (on three
subsequent days) with treatment times from about 7 and ten hours
per day using a detoxification system as described below.
No adverse reactions were observed during the treatment. Blood
pressure, oxygen saturation, blood glucose level and blood pH
improved slowly but continuously.
During the treatment the patient recovered from unconciousness and
fully awoke after the second treatment without remaining
neurological symptoms of encephalopathy (no flapping tremor, fully
orientated, no delay in communicational and physical response,
recovery of normal reflexes). During the following days the
activity of liver enzymes decreased slowly to 50%, the
thromboplastine time increased to 40%. Within three following days
the platelet count increased up to 130.times.10.sup.9 /liter and
antithrombine III to 43%.
______________________________________ Tab. 12. Patient 1: Total
serum bilirubin levels pre- and post-treatment Total serum
bilirubin time of before after Total No. of treatment treatment
decrease treatment (h) (mg/dl) (mg/dl) (mg/dl)
______________________________________ 1 7 36 25.3 10.7 2 10 34.5
23.2 11.3 3 8 23.5 17.5 6.1
______________________________________
______________________________________ Tab. 13. Patient 1:
Unconjugated serum bilirubin levels pre- and post-treatment Total
serum bilirubin time of before after Total No. of treatment
treatment decrease treatment (h) (mg/dl) (mg/dl) (mg/dl)
______________________________________ 1 7 3.0 2.5 0.5 2 10 3.3 2.3
1.0 3 8 1.8 1.7 0.1 ______________________________________
Patient 2
male, 34 years old, chronic alcoholic liver disease
The patient (120 kg bodyweight) was known as alcoholic for six
years and admitted to the hospital for slowly increasing jaundice
accompanied by signs of infection (fever, high white blood cell
counts, left shift), loss of appetite and impairment of general
status. He was treated for three weeks conservatively. As the
general status worsened rapidly under conservative treatment and
serum bilirubin concentrations rose to levels up to 37.7 mg/100 ml
the patient was treated with the MARS method. During five days of
treatment no further worsening of the general status of the patient
did occur. Treatment time of four hours obviously was too short for
the patient. On day 4 a short 2 hour MARS treatment was combined
with a two hours hemodiafiltration. The very low total bilirubin
difference of that day is probably a combined effect of ineffective
treatment and sufficient hemofiltration. However, even the short
treatment time allowed bilirubin separation as well as separation
of hydrophilic uremic toxins like urea, creatinine or ammonia (due
to the second dialyzer enclosed in the dialysate circuit).
______________________________________ Tab. 14. Patient 2: Total
bilirubin levels pre- and post- treatment Total serum bilirubin
time of before after Total No. of treatment treatment decrease
treatment (h) (mg/dl) (mg/dl) (mg/dl)
______________________________________ 1 4 37.7 32.2 5.5 2 5 37.0
29.1 7.9 3 4 32.4 27.5 4.9 4 2 .degree. 2 32.8 32.1 0.7 (MARS +
HDF) 5 4 32.4 28.5 3.9 ______________________________________
______________________________________ Tab. 15. Patient 2: Serum
urea and creatinine levels pre- and post-treatment No time Urea
(mg/dl) Creatinine of of before after before after treatment
treatment treatment ______________________________________ 1 4 160
138 3.9 3.1 2 5 194 153 4.1 3.4 3 4 208 152 4.7 3.6 4 2 + 2 226 136
5.2 3.1 (Mars + HDF) 5 4 202 148 4.9 3.7
______________________________________
Patient 3
female, 29 years old, chronic alcoholic liver insufficiency
The patient had a long history of alcohol abuse with a known
chronic liver insufficiency that was clinically estimated to be on
a precirrhotic level. Admittance happened upon increasing jaundice
and worsening of the general status. Under conventional treatment
the patient went into hepatic encephalopathy grade IV, i.e. she
fell into deep coma and did not respond to painful stimuli.
Bilirubin levels rose to 29,5 mg/100 ml. She showed
life-threatening severe acid-base disturbances, pulmonary edema,
hypoxemia on the first day of MARS- treatment and was estimated
moribund by experienced medical doctors. In this status treatment
with the method according to the present invention was started as
ultima ratio. Within two days the severe life-threatening status
totally changed into a moderate state of disease with normal
acid-base values, normooxygenation and normal ventilation. The
patient awoke from coma and went back to encephalopathy grade I/II,
i.e. she reacted adequately although with a slurred speech. The
patient was treated for six consecutive days for six hours per day.
The bilirubin level continuously decreased to 16,2 mg/100 ml during
this time.
______________________________________ Tab. 16. Patient 3: Total
serum bilirubin levels pre- and post-treatment during 4 MARS
sessions (1-4) and two hemodiafiltration sessions (5-6) Total serum
bilirubin time of before after Total No. of treatment treatment
decrease treatment (h) (mg/dl) (mg/dl) (mg/dl)
______________________________________ 1 5 29.0 24.3 4.7 2 6 26.9
19.9 7.0 3 5 23.4 17.8 5.6 4 5 18.3 13.1 5.2 5 5 15.9 12.9 3.0 6
4.5 16.5 15.6 0.9 ______________________________________
Patient 4
female, 41 years old, digoxin intoxication
The patient showed typical clinical signs of digoxin intoxication
such as tachyarrhytmia, loss of appetite, vomitting, diarrhoea; ECG
showed ventricular arrhythmia and typical ST-changes. After four
hours of treatment the clinical symptoms normalized completely and
so did the ECG changes.
Tab. 16 shows single pass clearances at the start and at the end of
the treatment. A highly toxic starting level and sufficient
regeneration of digoxin by on line-adsorption/dialysis in the
closed-loop liquid (B) dialysate circuit (3,95 to 0,5 and 2,55 to
0,54) were noted. No saturation of the liquid (B) dialysate circuit
and the adsorbents (PBS) was recognized. Since digoxin is
distributed in many tissues the blood level normally decreases very
slowly because of continuous influx from the other tissues.
Therefore a longer treatment, ideally a continuous one, would be
desirable.
______________________________________ Tab. 17. Patient 4: Single
pass clearances of digoxin during 4 hours of MARS digoxin serum
dialysate time location concentration (.mu.mol/l)
______________________________________ start pre 7.45 0.50 dialyzer
post 3.66 3.95 dialyzer end pre 5.84 0.54 dialyzer post 3.86 2.55
dialyzer ______________________________________
The above is intended to illustrate the present invention but is
not limiting. Numerous variations and modifications can be effected
without departing from the spirit and scope of the novel concepts
of the invention. It is to be understood that no limitation with
respect to the specific compositions and uses described herein is
intended or should be inferred.
* * * * *